Title : Simultaneous quantification of estrogens and glucocorticoids in human adipose tissue by liquid-chromatography-tandem mass spectrometry Running Title : Extraction of steroid hormones in adipose tissue

The presence of estrogens, androgens and glucocorticoids as well as their receptors and steroid converting enzymes in adipose tissue has been established. Their contribution to diseases such as obesity, diabetes and hormone-dependent cancers is an active area of research. Our objective was to develop a LC-MS/MS method to quantify bioactive estrogens and glucocorticoids simultaneously in human adipose tissue. Estrogens and glucocorticoids were extracted from adipose tissue samples using solid-phase extraction. Estrogens were derivatized using 1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine (PPZ) and methyl iodide to generate a permanently charged molecule (MPPZ). Steroids were separated and quantified by LC-MS/MS. The limit of quantitation for the steroids was between 15 and 100 pg per sample. Accuracy and precision were acceptable (< 20%). Using this method, estradiol, estrone, cortisone and cortisol were quantified in adipose tissue from women with and without breast cancer. This novel assay of estrogens and glucocorticoids by LC-MS/MS coupled with derivatization allowed simultaneous quantification of a panel of steroids in human adipose tissue across the endogenous range of concentrations encountered in health and disease. Abbreviations: C3-E1, 2,3,4-[ C3] estrone; C3-E2, 2,3,4-[ C3] 17β-estradiol; APCI, Atmospheric pressure chemical ionization; DCM, Dichloromethane; E, Cortisone; E1, Estrone; E2, Estradiol; ESI, Electrospray ionization; Et2O, Diethyl ether; EtOAc, Ethyl acetate; EtOH, Ethanol; D4F, 9,12,12,12 [ H4]-cortisol; FA, Formic acid; GC-MS/MS, Gas chromatographytandem mass spectrometry; HPLC, High-performance liquid chromatography; IS, Internal standards; LC-MS/MS, Liquid chromatography-tandem mass spectrometry; LLE, Liquid-liquid extraction; LOQ, Limit of quantitation; LOD, Limit of detection; MeOH, Methanol; MPPZ, 1(2,4-dinitro-5-fluorphenyl)-4,4-dimethylpiperazinium; MRM, Multiple reaction monitoring; MTBE, methyl t-butyl ether; OFN, Oxygen-free nitrogen; PPZ, 1-(2,4-dinitro-5-fluorphenyl)-4J ur na l P r -p ro of Laforest et al. 2019, page 5 methylpiperazine; RME, Relative mean error; RSD, Relative standard deviation; SNR, Signal-tonoise ratio; SPE, Solid-phase extraction; UHPLC, Ultra-high-performance liquid chromatography.